A. Field of the Invention
The invention relates, in one aspect, to a procedure for synthesis of well-defined conjugates of peptides covalently bonded to a tolerogenic polymer such as monomethoxypolyethylene glycol (MPEG) or polyvinyl alcohol (PVA). The first step in said synthesis involves succinylation of free-hydroxyl groups on the tolerogenic polymer by reaction with succinic anhydride. The polymer is then coupled to one or the other terminus, for instance via the carboxyl of a succinyl group to the .alpha.-NH.sub.2, of a peptide. This is achieved while maintaining intact all the side-chain protecting groups on the peptide. The MPEG or PVA-peptide conjugate are cleaved from a synthetic resin and purified. This method results in the preparation of conjugates in which one molecule of tolerogenic polymer is specifically coupled to one or the other or both of the termini of an otherwise unaltered peptide molecule.
In order to test the ability of such tolerogenic peptides to suppress antibody responses in an autoimmune disease, a synthetic peptide, .alpha.125-148, corresponding to a myasthenogenic region of Torpedo californica acetylcholine receptor (AChR) was conjugated to monomethoxypolyethylene glycol (MPEG). Injection of mice with the MPEG-(.alpha.125-148) conjugate and subsequent immunization with whole Torpedo AChR suppressed the development of experimental autoimmune myasthenia gravis (EAMG) by electrophysiological criteria. In anti-AChR antisera from these animals, the antibody response against the unconjugated peptide .alpha.125-148 was decreased while the antibody responses against whole AChR and other epitopes were not altered. There were no detectable changes in T cell proliferation responses to peptide .alpha.125-148 or to whole AChR in these animals. Prior injections with a "nonsense" peptide mPEG conjugate had no effect on responses to the subsequent immunization with whole Torpedo AChR. The results indicate that the mPEG-(.alpha.125-148) conjugate has epitope-specific tolerogenicity for antibody responses in EAMG, and that the region .alpha.125-148 plays an important pathophysiological role in EAMG. These studies strongly indicate that other epitope-directed tolerogenic conjugates will be useful for future immunotherapies of human myasthenia gravis.
Tolerogenic peptides are also disclosed for diseases as diverse as ragweed pollen allergy and Grave's disease. The strategy of specific suppression of the antibody response to a pre-determined epitope using a synthetic mPEG-peptide conjugate will be useful in manipulation and suppression of unwanted immune responses such as autoimmunity and allergy.
B. Description of the Related Art
Some of the earliest methods combining the use of amino acid synthesis with polymers such as polyethylene glycol (PEG) were as a resin for the synthesis of peptides. These methods relate generally to attachment of PEG at the carboxy terminus of the growing peptide chain. Typically, the resulting synthetic peptide is ultimately cleaved from the PEG resin and purified. For instance, Anzinger and Mutter (1982), relates to modified PEG moieties capable of binding both C- and N-termini of synthetic peptides for purposes as a soluble carrier and as a solubilizing protecting group in peptide syntheses.
Alternatively, tolerogenic polymers have been used to derivatize proteins and peptides in a permanent, covalent fashion. Prior research has shown that mPEG-protein conjugates may be constructed by non-selective coupling of the polymer to proteins, usually via the e-amino groups of lysine residues on the surfaces of such proteins. Such substitutions result in multiple derivatives.
Previous studies, for instance, have shown that conjugation of polyethylene glycol, monomethoxypolyethylene glycol (MPEG: or polyvinyl alcohol (PVA) with various protein antigens causes a loss of most of the antigenicity of the native antigens (Abuchowski et al., 1977; Lee and Sehon 1977, 1978a; King et al., 1977, 1979; Davis et al., 1980; Sehon and Lang, 1986). It has also been demonstrated that prior injection of animals with antigen-mPEG conjugates leads to the development of tolerance to subsequent immunization with the native antigen (Lee and Sehon 1977, 1978b; King et al., 1979).
mPEG-derivatization has been used to produce whole-protein and protein fraction conjugates. Abuchowski et al. (1977) relates, for instance to the derivatization of bovine serum albumin with mPEG causing this molecule to become essentially non-immunogenic. Lee and Sehon (1977) similarly converted ovalbumin and mixtures of non-dialyzable allergenic constituents of the aqueous extract of ragweed pollen. King et al. (1979) relates to the comparative study of different tolerogenic polymers indiscriminately conjugated with ragweed antigen E. Nishimura, et al. (1983), relates to the use of an indiscriminately PEG-derivatized snake venom with a molecular weight of 36,000. British Patent 1 469 472 appears to relate to the desire to provide polypeptides such as insulin a longer residence time in the circulatory system and a lessened allergic reaction in the same by apparently indiscriminate conjugation of such polypeptides to PEG.
In certain cases, the indiscriminate conjugation of polymer to protein has been controlled to a limited extent. For instance, European Patent Application 0 335 423 relates to the hG-CSF polypeptide derivatized with a PEG moiety. The derivatization appears to be indiscriminate even though the disclosure does provide for as few as a single PEG molecule per molecule of hG-CSF by controlling the stoichiometric ratios of the polymer to protein. Kita, et al. (1990), relates to the selective modification of one of three (including the N-terminal) residues within human interferon in order to obtain active interferon from recombinant bacteria. The PEG modification was seen to increase the serum half-life of the interferon without substantial decrease in its biological efficacy.
Tolerogenic polymers have also been used to derivatize certain peptides. In the past, modifying peptides with PEG generally required use of methods for activation including: (1) activation with triazine derivatives of PEG; (2) activation of PEG using the active ester method with N-hydroxylsuccinimide; (3) activation of PEG with carbonyldiimidazole; (4) activation of PEG with aldehydes; and so on. These modification methods involve modifying the amino groups at the N-terminal or in the side chain of the lysine residues of the peptides. For instance, Becker and Bayer (1979), relates to synthetic peptides with PEG conjugated to NH.sub.2 groups which are available for coupling. In certain cases, where the only reactive NH.sub.2 group in the peptide is the .alpha.-NH.sub.2, the PEG molecule was conjugated to the N-terminus of the peptide. Such methodology, however, relied on the fact that no other reactive amine groups existed in the peptide to be derivatized.
In some instances, peptides have been derivatized using distinct chemical moieties apart from the N-terminus amino group. Ueyama, et al. (1985), relates to the conjugation of PEG to cysteine-containing peptides through the carboxy groups in said cysteines. European Patent Applications 0 340 741 and 0 400 486 relate to PEG derivatives for use as a peptide (particularly protein)-modifying reagent in peptides having guanidino groups. PCT International Application, Pub. No. 90/12874, relates to the modification of polypeptides such as IL, G-CSF or EPO by non-N-terminal conjugation of PEG to cysteine residues in such polypeptides. Sartore, et al. (1991), relates to a method of producing a reagent comprising mPEG attached to an amino acid or a peptide, the amino acid or peptide functioning as a traceable spacer arm between the reagent and a derivatizable polypeptide in order to change its immunological properties. The attachment of the mPEG polymer to the peptide was at the carboxy terminus leaving, apparently, a free reactive amine functional group at the other terminus.
It is known, however, that heterogenous mixtures of "PEGylated" polypeptides and peptides are unsuited for pharmacological purposes (see e.g., PCT International Application, Pub. No. 90/12874). Indiscriminately conjugated proteins and peptides will, almost invariably, be expected to contain a mixture of molecular species or derivatives.
The use of tolerogenic antigens, alloantigens and allergens has received recent interest from the medical community for the treatment of autoimmune type disease. Typically, as noted above, the methods of the prior art utilize randomly derivatized whole antigen. Certain models of these diseases are known, however, which may serve as a testing ground for new approaches.
For instance, animals immunized with acetylcholine receptors (AChRs) in the presence of complete Freund's adjuvant produce autoantibodies against AChRs and develop a neuromuscular disease similar to human myasthenia gravis. In this experimental animal disease, called experimental autoimmune myasthenia gravis (EAMG), the majority of the autoantibodies is directed against the main extracellular part of the a subunit of AChR. The mapping of the complete antibody recognition profile, using overlapping synthetic peptides representing the entire extracellular part of the .alpha. subunit of Torpedo californica AChR, demonstrated that the peptide .alpha.125-138 contains a major antigenic site (Mulac-Jericevic et al., 1987). This epitope is located within the sequence .alpha.125-148 which is a potent region for induction of EAMG (Lennon et al., 1985) and contains the acetylcholine binding site (McCormick and Atassi 1984).
Other autoimmune diseases and other undesirable immune responses such as allergic responses have been investigated sufficiently well to identify similar specific epitopes which may be the principal causative agent in the disease. Thus, for instance, ragweed pollen allergy is a condition resulting from IgE responses to ragweed allergens such as antigen E, antigen K and Ra3. Thus, it is known from the work of the present inventors that one can map the IgG and IgE antibody and the T-cell epitopes of Ra3 (Atassi and Atassi, 1985, 1986; Kurasaki et al. 1986). Animal models (rat) exist which are used to study the allergic responses.
Similarly, Grave's disease is an autoimmune disease caused by antibody and T-cell responses to epitopes on thyroid-stimulating receptor (TSHR). Recently, the hormone-binding regions on TSHR were localized (Atassi, et al. 1991). As is known due to recent press coverage, both human (President and Mrs. George Bush) and animal (the family pet dog of the President and First Lady) forms of this disease are known.
Where mixtures of indiscriminately derivatized peptides; such as the specific epitopes described above are used as tolerogens, problems associated with reproducibility and efficacy are common. In particular, in cases where autoimmune disease are the result of limited specific epitopes being the target of the autoimmune antibodies, tolerogenic mixtures are not desirable. What are needed are specifically-derivatized, epitope-specific conjugated peptides.